Cbln1 regulates axon growth and guidance in multiple neural regions

The accurate construction of neural circuits requires the precise control of axon growth and guidance, which is regulated by multiple growth and guidance cues during early nervous system development. It is generally thought that the growth and guidance cues that control the major steps of axon development have been defined. Here, we describe cerebellin-1 (Cbln1) as a novel cue that controls diverse aspects of axon growth and guidance throughout the central nervous system (CNS) by experiments using mouse and chick embryos. Cbln1 has previously been shown to function in late neural development to influence synapse organization. Here, we find that Cbln1 has an essential role in early neural development. Cbln1 is expressed on the axons and growth cones of developing commissural neurons and functions in an autocrine manner to promote axon growth. Cbln1 is also expressed in intermediate target tissues and functions as an attractive guidance cue. We find that these functions of Cbln1 are mediated by neurexin-2 (Nrxn2), which functions as the Cbln1 receptor for axon growth and guidance. In addition to the developing spinal cord, we further show that Cbln1 functions in diverse parts of the CNS with major roles in cerebellar parallel fiber growth and retinal ganglion cell axon guidance. Despite the prevailing role of Cbln1 as a synaptic organizer, our study discovers a new and unexpected function for Cbln1 as a general axon growth and guidance cue throughout the nervous system.

2. Fig. 3F and 3H show the in vitro commissural axon cultures treated with GPN to block Cbln1 exocytosis resulting in slower axon growth rate. First, the image panels in 3F does not appear to be different in terms of illustrating axon growth rate. Second, did the investigators look at whether the lack of Cbln1 signaling in the axons, especially in the in vivo conditional knockout mutants, only slow down the growth rate and that the mutant axons will eventually catch-up. This would make sense as there are other attractant and growth promoting molecules used by the dorsal spinal commissural neurons, like Netrin-DCC.
For the first point, we are sorry for the confusion for the reviewer. There is no discussion or speculation as to why Cbln1-Neurexin 2 is required by the dorsal commissural axons or other neurons and their axons for promoting growth and guidance when there are already known attractants and growth factors doing the same job. Is Cbln1-Neurexin 2 required for a specific subset of commissural axons as there are at least 4 different types, dI1-dI4, in the dorsal rodent spinal cord during development. 3 We agree with the reviewer that it is surprising that Cbln1-Neurexin 2 which has been shown to work as synaptic organizers is also required by the dorsal commissural neurons and other neurons for promoting growth and guidance when there are already known attractants and growth factors doing the same job. However, quite a few studies have shown that these synaptic organizers are also involved in earlier neural development including axon development. In C. elegans, Nrxn promotes neurite outgrowth of DVB neurons [1], and a neurexin-related protein, BAM-2, regulates axonal branches [2]. In Drosophila, homolog of α-neurexin (DNrx) restricts axonal branching [3], and neurexin and neuroligin-based adhesion molecules regulate axonal arborization growth independent of synaptic activity [4]. These studies suggest that these synapse organizers are also required in early neural development to regulate axon development, working together with those already known attractants and growth factors.
Together, these findings suggest that both axon guidance cues & growth factors and synaptic organizers have dual roles in both earlier axon development and later synapse organization.
We thank the reviewer for asking if Cbln1 is only required for a specific subset of commissural axons as there are at least 4 different types, dI1-dI4, in the dorsal rodent spinal cord during development.
Co-immunostaining of Cbln1 with Lhx2, a dI1 DCN marker [80], confirmed the expression of Cbln1 in the dI1 DCNs of developing spinal cords from E10.5 to E12.5 (Fig 1B-1D). We continued to find that expression of Cbln1 was also detected in dI2-4 DCN subpopulations (revised S1D and S1E Fig). These data suggest that Cbln1 might regulate commissural axon development in all dI1-dI4 DCN neurons.
4. Finally, a minor correction is needed on Page 13 in the subsection "Cbln1-Nrxn signaling in axon development", first sentence: "esp." needs to be spelled out.
We thank the reviewer for pointing out this and have corrected it now. 1. At the beginning of the results, "differentially expressed genes" needs to be clarified by adding "over developmental time." As written now, it is quite vague. 5 We thank the reviewer for the suggestion, and we now have revised this in the manuscript.
For Fig. 1A, as suggested, we now have highlighted Cbln1 expression in motor neurons at E11.5 and E12.5.
3. Figure 1B  We thank the reviewer for the suggestion, and we now have shown the higher magnification insets to highlight the expression of Cbln1 in Lhx2 + dI1 DCN neurons (Fig. 1B'-1D').
4. Figure 3A-C: Old work from Tessier-Lavigne group showed no outgrowth of spinal explants, unless netrin was present. Was a modification to your system made? Commenting on this would be helpful.
Both the purpose and the design of our DCN explant culture experiments are different from those of Tessier-Lavigne group. We wanted to test if Cbln1 could regulate commissural axon growth.
However, Netrin1 could promote axon growth, which may cover the effect of Cbln1. So we could not add Netrin1 in our study. Tessier-Lavigne group used Netrin1 to coat plate and did the culture for 16 h [81]. We use collagen gel and culture the explants for 2 days: there are indeed few axons growing in 24 h; however, after 40 h, explants have lots of axons growing out.
3F-H: worth using an additional inhibitor to nail this down a role for the lysosome or otherwise more clearly citing role for lysosome exocytosis in cerebellin-1 secretion from other papers for those not seeped in the field.?
We thank the reviewer for the suggestion. It has been reported that Cbln1 co-localizes with the lysosomal enzymes cathepsin B and D in the adult mouse brain [82,83], indicating the lysosome may regulate Cbln1 secretion in commissural axons. To test this, we applied different lysosome inhibitors to the DCN cultures and checked their effects on Cbln1 secretion from commissural axons. In addition to GPN, we also used Bafilomycin A1 (BafA1), which blocks lysosomal functions through working as a specific inhibitor of vacuolar H + -ATPase [84]. Treatment of DCN cultures with GPN or BafA1, followed 6 by an IF protocol to detect surface Cbln1 by leaving out the permeabilization steps, showed loss of Cbln1 IF signals on the commissural axon surface (Fig 3F and 3G; revised S3B and S3C Fig), suggesting that Cbln1 is released from lysosomes in commissural axons and growth cones. 5. Figure 6F (now removed in the revised version): clarify in the text that this addition to wildtype, although would be interesting if rescued in knockout?
We thank the reviewer for the suggestion, and we now have performed the rescue experiment to replace the old data (original Fig. 6F is now removed in the revised version). Axon growth rates of Cbln1-deficient GCs in vitro were significantly decreased compared with control neurons (revised Fig   6D and 6E), suggesting that the cell-autonomous Cbln1 is required for GC axon growth. Similar to Cbln1 on commissural axons, extrinsic application of the recombinant hCbln1 (rhCbln1) protein to the GC axons could efficiently rescue this axon growth defect (revised Fig 6D and 6E).
6. Figure 7C: Tag1 is marking axons of RGC---how were the regions defined, as it is unclear.
Optic chiasm forms a specific shape [85,86], where Tag1 + RGC axons traverse the chiasm region and enter the optic tracts in the lateral wall of the diencephalon [87]. We used Tag1 to mark the RGC axons navigating through this part. We now have removed the lines highlighting Tag1 IF signals.
We now have included the representative images for the axon guidance assay using retinal explants (revised Fig. 7D), and the quantification data were also updated (revised Fig. 7E).
8. Figure 7H (Fig. 7I in the revised version): Why don't we see ipsilateral projection here? My understanding is that DiI labeling would also label the ipsilateral axons. This DiI tracing experiment was performed at E13 (revised Fig. 7I). Ipsilateral RGC neurogenesis in retina starts to increase at E13. It is hard to observe the ipsilateral axons at E13 since there are only a few axons which just reach the optic chiasm and start to turn to the ipsilateral side [88]. Our purpose is to check the guidance of contralateral RGCs (Brn3a + Nrxn2 + ), so we checked the optic chiasm at the beginning timepoint (E13), and continued to check the terminal projection in LGN at P4 (revised Fig.   7K and 7L). We thank the reviewer for suggesting these interesting experiments. We could not show the colocalization of Cbln1 and Nrxn2 at the axon/growth cone in the cultured neurons because both Cbln1 and Nrxn2 antibodies that we tried to get working in the lab are from rabbit. As suggested, we checked surface Cbln1 distribution after Nrxn2 knockdown in culture neurons. We found that axonal surface Cbln1 signals were decreased after Nrxn2 knockdown in culture DCN neurons (revised S5E and S5F Fig). * In the discussion, I would be interested in hearing the authors' thoughts on the other Cerebellin isoforms/any possible redundancy (or lack thereof) in axon guidance.
Cbln1 and Cbln2 bind to GluRδ2 and Nrxn1-3, while Cbln4 is a highly specific ligand for DCC [89]. In addition, Cbln4-null mice did not show any defect in commissural axon guidance in the developing spinal cord [89].
Cbln3 has only been studied in synapse structural and functional regulation. Cbln3 is secreted only when it is bound to Cbln1 [90]. Cbln1 and Cbln3 have been shown to reciprocally regulate each other's degradation and secretion, so that Cbln1 and Cbln3 proteins are both lost in Cbln1-null mice, while Cbln3-null mice lack Cbln3 but have a robust increase in Cbln1 protein level [90]. Consequently, Cbln1 and Cbln3 single knockout mice have different phenotypes while Cbln1-Cbln3 double mutant mice have deficits identical to those of Cbln1-null mice [90]. It would be interesting to check if Cbln3 is expressed in the developing dorsal spinal cord and brain, and if there is a similar Cbln1-Cbln3 interaction mechanism in the earlier embryonic stages.
It has been reported that Cbln1 and Cbln2 have redundancy in cerebellum but not in thalamic neurons in the adult mice [91], supporting a tissue-specific redundancy in adulthood. Cbln2 is expressed in the developing chick dorsal spinal cord [92], and our own in situ hybridization result also 8 shows Cbln2 expression in the developing mouse dorsal spinal cord (unpublished data). Cbln1-Cbln2 double cKO may be useful to check their redundancy in the developing brain regions and axon developmental processes described in our current study.